and the cells removed by centrifugation. These were resuspended in sterile 1949a), growth was measured in terms of acid production while dextran was

Transcription

1 THE NUTRITIONAL REQUIREMENTS OF LEUCONOSTOC DEXTRANICUM FOR GROWTH AND DEXTRAN SYNTHESIS1 VIRGINIA WHITESIDE-CARLSON AND CARMEN L. ROSANO Biochemistry Department, Medical College of Alabama, Birmingham, Alabama Received for publication June 18, 1951 The recent literature contains numerous references to the use of partially hydrolyzed dextrans as plasma substitutes (Bull et al., 1949; Thorsen, 1949). Because of this interest, and as part of a general study of bacterial polysaccharide synthesis, the nutritional requirements of the major dextran forming organisms, Leuconostoc mesenteroides and L. dextranicum, are being investigated (Whiteside- Carlson and Carlson, 1949a). The present report deals with Leuconostoc dextranicum, strain "elai," which is unique in causing essentially complete conversion of the glucose half of the sucrose molecule into dextran. EXPERIMENTAL METHODS Media. The basal medium employed, comprising aimno acids, purines, pyrimidines, vitamins, and inorganic salts, was essentially the same as that described in a previous publication (Whiteside-Carlson and Carlson, 1949a). The sodium acetate-ammonium chloride buffer was replaced by one containing sodium acetate and potassium acetate in the amounts of 20 g and 5 g per liter, respectively, since it was observed that ammonium ion was detrimental to the growth of Leuconostoc. Vitamin B12 was used at a level of mg per liter; pyridoxine, pyridoxal, and pyridoxamine were each added at a concentration of 0.2 mg per liter. Sucrose was present in a concentration of 100 g per liter, while glucose or fructose was used at half this level. Inocula. The stock culture was carried in a 2 per cent sucrose nutrient agar. It was transferred to tubes of the synthetic amino acid medium complete except for biotin, pyridoxal, and pyridoxamine, incubated 24 hours at room temperature, and the cells removed by centrifugation. These were resuspended in sterile saline and again separated by centrifugation, following which they were diluted in sterile saline to a point giving readings of approximately 95 per cent light transmission as measured by the Klett-Summerson colorimeter. One drop of these suspensions was added to each 3-ml tube of experimental medium. The incubation temperature was 25 C. Analytical methods. As described previously (Whiteside-Carlson and Carlson, 1949a), growth was measured in terms of acid production while dextran was determined by a gravimetric procedure, both measurements being made in triplicate. I This work was supported in part by a grant from the Division of Research Grants and Fellowships of the National Institutes of Health, United States Public Health Service. 583

2 584 VIRGINIA WHITESIDE-CARLSON AND C. L. ROSANO [VOL. 62 RESULTS AND DISCUSSION In table 1 are presented data for the effect of the omission of single vitamins on acid production in glucose, fructose, and sucrose media, and on dextran yields from sucrose. In all three carbohydrate media nicotinic acid, thiamin, and pantothenic acid were found to be highly essential for growth as measured by acid production. With no more than the trace growth obtained in media deficient in nicotinic acid or pantothenic acid, however, the dextran yields were still nearly half of the theoretical value. The relatively low level of growth obtained in the thiamine deficient medium was accompanied by synthesis of almost the theoretical yield of the polysaccharide. As a control, tubes of sucrose TABLE 1 Effect of vitamin omission on acid production in glucose, fructose, or sucrose media and on dextran yield in a sucrose medium (72 hours' incubation time) ACID PtODUCTION ML 0.01 N NaOH DEXTRAN VITAMIN OMIITTED PROM MEtDIUM PER - XL MEDIUM - YIEWLD (FItOM Glucose Fructose Sucrose SUCROSE) ml ml ml % None Nicotinic acid Thiamin Pantothenic acid Riboflavin Pyridoxine, pyridoxal, pyridoxamine Pyridoxine Pyridoxal Pyridoxamine p-aminobenzoic acid Folio Acid Biotin Vitamin B * As per cent of theoretical yield. medium containing no added vitamins were inoculated. Dextran yields in such tubes were found to average less than 4 per cent of theory. From these and other studies, it is apparent that there is no correlation between growth, as measured by acid production, and dextran yield. Indeed, as was reported previously (Whiteside-Carlson and Carlson, 1949a,b) polysaccharide yields for other strains of Leuconostoc can be increased by employing conditions which somewhat limit the ability of the organisms to ferment sucrose. In addition to the requirement for the three vitamins listed previously, L. dextranicum also showed a requirement for biotin in media containing glucose or fructose, but not in sucrose. This result is in accord with previously published findings using Leuconostoc (Carlson and Whiteside-Carlson, 1949). The foregoing results were extended by investigating the effect of vitamins added singly to a basal medium containing the essential vitamins, nicotinic

3 1951] NUTRITIONAL REQUIREMENTS OF L. DEXTRANICUM 585 acid, pantothenic acid, and thiamin. The results obtained showed that the addition of no single vitamin resulted in stimulation of growth equivalent to that observed in the complete media. Folic acid, PAB, and pyridoxal or pyridoxamine were most effective as growth stimulants and hence appeared to constitute the second most essential group of vitamins. Biotin, even though the results recorded in table 1 indicated it to be second in importance only to nicotinic acids, thiamin, and pantothenic acid, failed to stimulate growth in the basal medium containing these vtamins. In the sucrose media, dextran production in all cases approached the theoretical limit regardless of the growth level attained. TABLE 2 Attempts to determine the minimum vitamin requirements of Leuconostoc dextranicum (96 hours' incubation time) FRUCTOSE SUCROSE VITAMINS ADDED TO MDIUM Acid R Acid frd*sugar final f,d* sugar fial, yieldra fre* conct fre* conct ml mg/ml m mg/m % None Completell Pyridoxamine p-aminobenzoic acid Pyridoxamine + PAB Pyridoxamine + biotin Pyridoxamine Pyridoxamine + B Pyridoxamine + B12, riboflavin + biotin Pyridoxamine + PAB biotin PAB + biotin PAB + riboflavin PAB + B Riboflavin + biotin + B * As ml 0.01 N NaOH per ml medium. t Initial fructose concentration = 48 mg/ml. t Initial sucrose concentration = 108 mg/ml; final sucrose concentration = 0 mg/ml in all cases. Nicotinic acid, pantothenic acid, and thiamin present in basal medium. 11 Includes all vitamins used in experiment. In view of these results, the experiments summarized in table 2 were conducted. To the basal medium containing the three essential vitamins were added various combinations of the other vitamins. Since the results recorded in table 1 showed that normal growth was obtained in the absence of folic acid as long as PAB was present, it was felt that the former vitamin could be omitted from further consideration. Likewise, of the B6 group only pyridoxamine was employed in these experiments. In the sucrose medium, a combination of pyridoxamine and PAB afforded nearly maximal growth. In the fructose medium, a combination of pyridoxamine, PAB, and biotin was required to achieve this result. Also il

4 586 VIRGINIA WHITESIDE-CARLSON AND C. L. ROSANO [vol. 62 included in table 2 are data on substrate utilization in the various media. Although not shown in the table, corresponding results were obtained in a glucose medium. These data again point up the apparent difference between utilization by L. dextranicum of preformed fructose as compared with fructose released during polymerization of the glucose half of the sucrose molecule into dextran. In some manner a biotin-requiring step in the utilization of fructose appears to be by-passed when fructose is made available during the polymeric degradation of sucrose. The alternative would seem to require the presence in the sucrose TABLE 3 Effect of omission of individual amino acids on acid production in glucose, fructose, or sucrose media and on dextran production in sucrose medium (96 hours' incubation time) AJ0NO ACID 0MI D FROM MDIM i&l 0.01 N NaOH PEt mi udm DEXTRN Glucose Fructose Sucrose Sucrose ml ml ml N L-Glutamic acid DL-Valine DL-Isoleucine L-Leucine DL-Alanine DL-MethiOnine L-Cysteine DL-Aspartic acid O DL-Lysine DL-Threornne DL-Phenylalanine DL-Tryptophan DL-Serine glycine L-Arginine L-Histidine L-Tyrosine L-Proline None sample used of a biotin complex, or of a biotin substitute, not capable of combination with avidin (Carlson and Whiteside-Carlson, 1949). The effects resulting from omission of the various purines and pyrimidines also were studied. The compounds used included adenine, xanthine, hypoxanthine, guanine, thymine, and uracil. In none of the three sugar media did omission of any single purine or pyrimidine result in significant lowering of growth or dextran production. Even omission of all these compounds still allowed essentially maximum growth and dextran synthesis. The experiment was repeated with the difference that only the basic vitamins were employed in the media. Under these more stringent conditions, however, the organisms were still found capable of satisfying their purine and pyrimidine requirements.

5 1951] NUTRITIONAL REQUIREMENTS OF L. DEXTRANICUM 587 The amino acid requirements of the organism were determined in glucose, fructose, and sucrose media. By omission of the various acids singly it was found (table 3) that glutamic acid, valine, threonine, tryptophan, and histidine were essential to growth and dextran production. The requirement for threonine in the sucrose medium appeared to be definitely less than that observed in the TABLE 4 Effect of phosphate ion concentration on growth and dextran synthesis (96 hours' incubation time) PO 4 PER M..EDum la 0.01 N NaOH PER ML MEDIUM DEXTIN Glucose Fructose Sucrose Sucrose mg ml ml ml % None TABLE 5 Effect of various minerals on acid production and dextran synthesis (96 hours' incubation time) MINERALS ADDED TO MEDIUM* 3m 0.01 N NaOH PER iml M:DIM DEXTRAN Glucose Fructose Sucrose Sucrose m ml ml % FeSO MnSO4 MgSO4 FeSO MnSO MgSO None * Concentration of minerals per L medium: FeSO4, 30 mg; MnSO4, 30 mg; MgSO4, 300 mg. NaCl, 300 mg per L was present in the basal medium. glucose and fructose media. Furthermore, omission of cysteine had little influence on growth in the sucrose medium, but was definitely inhibitory in media containing the constituent monosaccharides. This result was obtained consistently in repeated experiments. The entire experiment also was repeated under conditions such that only the basic essential vitamins were present, and with no added purines or pyrimidines. The same amino acid requirements were

6 588 VIRGINIA WHITESIDE-CARLSON AND C. L. ROSANO found as existed in the case of the complete media. In neither type of experiment was any instance noted in which an amino acid was more essential for dextran synthesis than for growth. Neither was the converse situation found, although the requirement for threonine tended in this direction. In tables 4 and 5 are summarized data on certain of the mineral requirements of the organism in the various carbohydrate media. In all three sugars approximately maximum acid production was reached at a phosphate concentration of 0.06 mg per ml medium, while dextran production reached its highest level at a concentration of mg per ml. From studies on preparations of the dextran synthesizing enzyme, Hehre (1943) has reported that phosphate ion is not directly involved in formation of the polysaccharide. Of the other inorganic additives studied, only manganous ion was found essential for full acid production (table 5). The trace of manganese present in the other medium constituents allowed sufficient growth for dextran production to approach the theoretical limit in the medium containing no added manganous ions. Culture conditions affording maximum acid production often were accompanied by slightly decreased dextran yields. Similar results were obtained in the studies on the vitamin requirements of the organism. SUMMARY [VOL. 62 The amino acid, vitamin, purine and pyrimidine, and mineral requirements of Leuconostoc dextranicum, strain "elai," for growth in sucrose, glucose, and fructose media were determined. In no instance was a nutrient found to be more essential for dextran synthesis than for growth in sucrose media. Amino acids essential for growth included glutamic acid, valine, threonine, tryptophan, and histidine. The omission of cysteine had little effect on growth in the sucrose medium but was definitely inhibitory in media containing the constituent monosaccharides. Nicotinic acid, thiamin, and pantothenic acid were found to be highly essential for growth in all three carbohydrate media. In sucrose media containing only these essential vitamins, full growth was not observed until either folic acid or p-aminobenzoic acid, and either pyridoxal or pyridoxamine, were added. In media containing glucose or fructose a similar situation held except that here the presence of biotin was also required. The organism showed essentially complete growth in the absence of any added purines or pyrimidines. Among the mineral elements tested definite requirements were shown only for manganous ions and phosphate ions. No correlation was observed between growth, as measured by acid production, and dextran yield. REFERENCES BULL, J. P., RICKETTS, C., SQUIRE, J. R., MAYCOCK, W. D' A., SPOONER, S. J. L., MOLLISON, P. L., AND PATERSON, J. C. S Dextran as a plasma substitute. Lancet, 256,

7 1951] NUTRITIONAL REQUIREMENTS OF L. DEXTRANICUM 589 CARLSON, W. W., AND WHITESIDE-CARLSON, V Biotin-carbohydrate interrelationships in the metabolism of Leuconostoc. Proc. Soc. Exptl. Biol. Med., 71, HEHRE, E. J Comparison of dextran synthesis by Leuconostoc enzyme with starch synthesis by potato phosphorylase. Proc. Soc. Exptl. Biol. Med., 64, THORSEN, G Dextran as a plasma substitute. Lancet, 256, WHITESIDE-CARLSON, V., AND CARLSON, W. W. 1949a The vitamin requirement of Leuconostoc for dextran synthesis. J. Bact., 58, WHITESIDE-CARLSON, V., AND CARLSON, W. W. 1949b Studies of the effect of paraaminobenzoic acid, folic acid, and sulfanilamide on dextran synthesis by Leuconostoc. J. Bact., 58,